ABSTRACTAlzheimer's disease and frontotemporal dementia are amongst the most common forms of dementia characterized by the formation and deposition of abnormal TAU in the brain. In order to develop a translational human TAU aggregation model suitable for screening, we transduced TAU harboring the pro-aggregating P301L mutation into control hiPSC-derived neural progenitor cells followed by differentiation into cortical neurons. TAU aggregation and phosphorylation was quantified using AlphaLISA technology. Although no spontaneous aggregation was observed upon expressing TAU-P301L in neurons, seeding with preformed aggregates consisting of the TAU-microtubule binding repeat domain triggered robust TAU aggregation and hyperphosphorylation already after 2 weeks, without affecting general cell health. To validate our model, activity of two autophagy inducers was tested. Both rapamycin and trehalose significantly reduced TAU aggregation levels suggesting that iPSC-derived neurons allow for the generation of a biologically relevant human Tauopathy model, highly suitable to screen for compounds that modulate TAU aggregation.

pone.0146127.g002: AlphaLISA optimizations on human brain extracts for total TAU, TAU aggregation and phosphorylation.(A, B) AlphaLISA on 2 different AD brain extracts show high hTAU10 (A) and AT8 (B) TAU aggregation signals compared to control brain samples. (C, D) AT8/hTAU10 (C) AlphaLISA on these AD brain extracts reveals high levels of phosphorylated TAU compared to control brain samples while both AD and control brain extracts display high HT7/hTAU10 (D) levels. Decreasing signals with increasing dilutions suggest no hooking of the samples. Representative curve of 1 experiment with 2 technical replicates is shown as RFU (relative fluorescence units) ± SD. (E, F) Western Blot on soluble (S) and insoluble (IS) fractions of control and AD brain extracts after Sarkosyl extraction shows HT7-positive (E) and AT8-positive (F) bands only in the Sarkosyl insoluble pellets of both AD patients, confirming the presence of TAU aggregates. M represents Magic Marker (band sizes) and T represents TAU ladder with all 6 TAU isoforms. All experiments have been confirmed at least twice.

Mentions:
Human brain extracts from 2 AD patients and 1 healthy control (Newcastle Brain Tissue Resource, Newcastle University; S1 Table) were obtained for extended validation of these TAU-quantification assays in a human setting. Brain extracts from both AD donors show high hTAU10 and AT8 TAU-aggregation signals (Fig 2A and 2B) as well as high TAU phosphorylation levels (Fig 2C) in comparison to control brain extract, while total TAU is expressed to a similar level in all samples tested (Fig 2D). Additionally, after Sarkosyl fractionation of both control and AD brain extracts, only the Sarkosyl insoluble fractions of the two AD brains reveal HT7-positive and AT8-positive bands (Fig 2E and 2F), confirming our AlphaLISA results.

pone.0146127.g002: AlphaLISA optimizations on human brain extracts for total TAU, TAU aggregation and phosphorylation.(A, B) AlphaLISA on 2 different AD brain extracts show high hTAU10 (A) and AT8 (B) TAU aggregation signals compared to control brain samples. (C, D) AT8/hTAU10 (C) AlphaLISA on these AD brain extracts reveals high levels of phosphorylated TAU compared to control brain samples while both AD and control brain extracts display high HT7/hTAU10 (D) levels. Decreasing signals with increasing dilutions suggest no hooking of the samples. Representative curve of 1 experiment with 2 technical replicates is shown as RFU (relative fluorescence units) ± SD. (E, F) Western Blot on soluble (S) and insoluble (IS) fractions of control and AD brain extracts after Sarkosyl extraction shows HT7-positive (E) and AT8-positive (F) bands only in the Sarkosyl insoluble pellets of both AD patients, confirming the presence of TAU aggregates. M represents Magic Marker (band sizes) and T represents TAU ladder with all 6 TAU isoforms. All experiments have been confirmed at least twice.

Mentions:
Human brain extracts from 2 AD patients and 1 healthy control (Newcastle Brain Tissue Resource, Newcastle University; S1 Table) were obtained for extended validation of these TAU-quantification assays in a human setting. Brain extracts from both AD donors show high hTAU10 and AT8 TAU-aggregation signals (Fig 2A and 2B) as well as high TAU phosphorylation levels (Fig 2C) in comparison to control brain extract, while total TAU is expressed to a similar level in all samples tested (Fig 2D). Additionally, after Sarkosyl fractionation of both control and AD brain extracts, only the Sarkosyl insoluble fractions of the two AD brains reveal HT7-positive and AT8-positive bands (Fig 2E and 2F), confirming our AlphaLISA results.

ABSTRACTAlzheimer's disease and frontotemporal dementia are amongst the most common forms of dementia characterized by the formation and deposition of abnormal TAU in the brain. In order to develop a translational human TAU aggregation model suitable for screening, we transduced TAU harboring the pro-aggregating P301L mutation into control hiPSC-derived neural progenitor cells followed by differentiation into cortical neurons. TAU aggregation and phosphorylation was quantified using AlphaLISA technology. Although no spontaneous aggregation was observed upon expressing TAU-P301L in neurons, seeding with preformed aggregates consisting of the TAU-microtubule binding repeat domain triggered robust TAU aggregation and hyperphosphorylation already after 2 weeks, without affecting general cell health. To validate our model, activity of two autophagy inducers was tested. Both rapamycin and trehalose significantly reduced TAU aggregation levels suggesting that iPSC-derived neurons allow for the generation of a biologically relevant human Tauopathy model, highly suitable to screen for compounds that modulate TAU aggregation.